As we are interested in studying the immune system in an intact animal, we use the laboratory mouse as an experimental model. To study the function of MHC molecules, we remove a gene from the germline of a mouse, so that it is missing in all cells of that mouse and in all cells of its progeny. Such knockout mice allow us to study the function of a gene in a whole animal. We have generated several knockout mice, which exhibit various defects in their immune system.

The first step in the presentation of antigens to MHC class I-restricted CD8 T cells is degradation of cytosolic protein antigens by the proteasome (Figure 1, left panel) (reviewed in 1, 2). The proteasome is a large proteolytic complex that contains many subunits, including two subunits, LMP2 and LMP7, encoded within the MHC locus. Peptides are then translocated across the endoplasmic reticulum (ER) membrane by the TAP peptide transporter. Peptides are then loaded onto the MHC class I molecule and transported to the cell surface for recognition by CD8 T cells. Recent studies have shown that MHC class I molecules are tethered to the TAP transporter via a novel protein, tapasin (3, 4). Research from Glen Grandea in the laboratory, while working in the laboratory of Thomas Spies at the Fred Hutchinson Cancer Research Center (Seattle, WA) showed that, in tapasin-deficient cell lines, assembly and surface expression of class I molecules are impaired (3). As Glen has recently generated tapasin knockout mice, he will be able to evaluate the in vivo role of tapasin in MHC class I-restricted antigen presentation, and in the intrathymic selection of CD8 T cells.

The assembly of MHC class II molecules is initiated in the ER by association of MHC class II a and b chains with the invariant chain (Figure 1, right panel) (reviewed in 5, 6). Invariant chain occupies the peptide-binding groove of the MHC class II heterodimer and, as such, avoids peptide binding. Invariant chain also functions to transport the class II heterodimer to the late endosomal compartments (MHC class II compartments or MIICs) where MHC class II molecules are loaded with peptide. Once in the MIIC, invariant chain is degraded by proteases (cathepsins) until only a small fragment, the class II-associated invariant chain peptide or CLIP region, remains associated with MHC class II. This CLIP fragment is then removed from MHC class II by the H2-DM peptide exchange factor (7). This enables antigenic peptides, generated from endocytosed proteins by cathepsins, to bind with MHC class II. To study the in vivo function of H2-DM we have generated H2-DM knockout mice (8). In cells from these animals, CLIP peptides cannot dissociate from MHC class II, and interaction with antigenic peptides is therefore inhibited. While overall cell surface expression of class II molecules in these mice was similar to wildtype, most of the class II heterodimers were bound by the CLIP peptide. Consequently, cells from these animals are deficient in the presentation of protein antigen to MHC class II-restricted T cells. Interestingly, cells from these animals are also poorly recognized by MHC class II-specific alloreactive CD4 T cells, indicating that peptide is a critical component for the recognition of MHC molecules by alloreactive T cells (9). Jean-Paul Kovalik in the laboratory is conducting studies to define the precise role of MHC-bound peptides in alloreactive T cell responses.

During their development in the thymus immature T cells undergo several selection processes (Figure 2) (10). These events are mediated by interaction of the T cell receptors (TCRs) expressed by immature T cells with MHC-peptide complexes expressed by epithelial and bone marrow-derived cells in the thymus. An immature T cell with TCRs that interact very strongly with self MHC-peptide complexes undergo apoptosis in a process termed negative selection. An immature T cell with TCRs that interact at an intermediate level with self MHC-peptide complexes receive a survival signal known as positive selection. Finally, immature T cells carrying TCRs with very weak affinity for self MHC-peptide complexes die by neglect. Thus, the overall strength or avidity of the interaction between the T cell and the selecting cells appears to be the critical factor that determines the fate of immature T cells.

One issue regarding T cell positive selection that remains controversial is the contribution of the MHC-bound peptides to this process (11). Since few MHC molecules reach the cell surface in an empty state (i.e. without peptide) positive selection occurs on MHC molecules that are bound by self peptides. This raises the possibility that the MHC-bound peptides may contribute to the specificity of this process and bias the mature T cell repertoire. The H2-DM deficient mice described above allowed us to test this issue directly. These animals represent a situation where nearly all MHC class II molecules are occupied by a single peptide, CLIP. We therefore investigated the T cell repertoire of these animals. Our results indicated that numbers of CD4 T cells in the peripheral lymphoid organs of these animals were significantly reduced, and that these cells had unusual reactivities (8). However, Nagendra Singh, currently in Medical College of Georgia, in the laboratory showed that, despite these defects, H2-DM knockout mice can generate CD4 T cell responses against a variety of antigens (12). Interestingly, we also found that these animals, when housed in conventional cages, spontaneously develop an inflammatory condition of the colon that resembles human inflammatory bowel disease. We are currently investigating the pathogenesis of the disease process in these animals.

Antigen-presenting Function of the MHC Class I-like Molecule CD1d

In addition to the well-studied MHC class I and class II molecules described above, many unusual or unconventional MHC molecules have been identified. One group of unusual MHC class I molecules is represented by the CD1 family of antigen-presenting molecules (reviewed in 13). Humans have 5 CD1 genes (CD1a, -b, -c, -d, and -e), whereas mice have 2 CD1 genes (CD1d1 and CD1d2) that are similar to the human CD1d gene. Unlike the conventional MHC class I and class II molecules that bind peptides, CD1 molecules bind lipids or glycolipids. For example, the human CD1a, -b and -c molecules can present mycobacterial lipid and glycolipid antigens to T cells (19). Similarly, the CD1d molecules of human and mouse can present glycolipid antigens such as phosphatidylinositol derivatives and glycosylceramides to an unusual group of T cells, termed natural killer T (NKT) cells (Figure 3) (13-15).

NKT cells express surface markers that are commonly found on conventional T cells and NK cells (Figure 3) (14, 15). These cells express a TCR composed of an invariant Va14-Ja281 chain together with a polyclonal Vb8 (and to some extent Vb7 or Vb2) chain. They also express typical NK cell markers, including members of the NK cell-activating receptor family (NKR-P1 molecules) and NK cell-inhibitory receptor family (Ly49 molecules). NKT cells also have a memory phenotype, expressing high levels of the CD69 and CD44 markers and low levels of the L-selectin marker. When activated, NKT cells quickly produce a variety of cytokines, including large amounts of IL-4 and significant amounts of IFN-g, and become cytotoxic. Although their physiological role remains unclear, NKT cells have been implicated in immune responses against pathogens, tumors, bone marrow grafts, and self antigens (14, 15).

Our laboratory has previously generated CD1d knockout mice to determine the role of CD1d in the development of NKT cells and to study the immunological function of NKT cells (16). CD1d knockout mice were largely deficient in NKT cells. Unlike wild-type animals, injection of anti-CD3 antibodies in CD1d knockout mice did not result in the production of large amounts of IL-4. Despite these defects, however, we showed that these animals, when immunized with model antigens in adjuvant, generate relatively normal T cell and antibody responses (15). Seokmann Hong in the laboratory is now evaluating the role of NKT cells in murine models of autoimmune disease, including diabetes in the non-obese diabetic (NOD) mouse and experimental allergic encephalomyelitis in SJL and PL/J mice, an experimental model for multiple sclerosis.

The glycolipid a-galactosylceramide (a-GalCer), originally isolated at Kirin Brewery Co (Japan) from marine sponges as an agent with profound antitumor activities in mice, can bind with CD1d and selectively activate NKT cells (15). We are conducting collaborative studies with the laboratory of Dr. Yasuhiko Koezuka at Kirin Brewery Co to evaluate the effects of this agent on adaptive immune responses in vivo. Nagendra Singh and Seokmann Hong in the laboratory showed that a-GalCer polarizes adaptive immune responses towards Th2-dominant immunity (17). Further, Seokmann Hong in our laboratory and Isao Serizawa at Kirin Brewery Co showed that this agent can inhibit diabetes in the NOD mouse. These findings indicated that a-GalCer may be useful for modulating immune responses during prophylaxis and therapy.